The enzymatic digestion of isolated DNA from procaryotes, eucaryotes, and plasmids is a time-consuming but necessary step for further gene studies and requires consistent cleavage of DNA molecules or segments at specific sites. Several factors can affect the outcome. Variations in yield and quality of the cleaved fragments from digestion to digestion probably result from inconsistent temperatures and enzymatic activity that occur over the hours it may take to effect complete digestion of large macromolecules. Impurities in DNA samples may retard processing and produce inconsistent products.
Traditionally, digestions of DNA with restriction endonucleases are carried out in a buffer containing salts and reducing agent in an incubator at 37.degree. C., considered to be the optimum temperature for activity of most enzymes. Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). The incubation time varies with the type and quality of DNA and the particular restriction endonuclease used. Although the digestion of plasmid DNA with some enzymes can be achieved in 1 hour using a large amount of enzyme, in general, cleaving genomic DNA with restriction endonucleases takes from eight hours to overnight and thus is a time-consuming technique by conventional methods. Accordingly, there remains a need for a rapid, efficient, and consistent procedure for digesting DNA.
The effects of microwave energy on chemical processes has been studied for many years. Abramovitch, et al., Org. Prep. Proced. Int. (1991) 23:683-711. Microwaves have been used to drive the synthesis of radiopharmaceuticals (Hwang, et al., J. Chem. Soc., Chem. Commun. (1987) 23:1799-801), hydrolyses (Abramovitch, et al., (1991) Tetrahedron Let. 32:5251-54), polymerizations (U.S. Pat. No. 3,432,413), and the synthesis of organic compounds (Gedye, et al., Can. J. Chem. (1991) 69:706-11; Ito, et al. J. Mol. Evol. (1990) 31:187-94). Hydrolysis of biological materials has also been studied. Jahngen, et al., J. Org. Chem. (1990) 55:3406-9; Margolis, et al., J. Auto. Chem. (1991) 13:93-95; Feinberg, et al. (1991) Analusis 19:47-55.
However, when living systems or tissues are exposed to concentrated microwave energy, the effects have been deleterious for the most part. Belkhode, et al., J. Microwave Power (1974) 9:23-29; Galli, et al., Meth. Enzymology (1982) 86:635-642; Huai, et al., J. Bioelectricity (1984) 3:361-366; Baranski S., Amer. J. Phys. Medicine (1972) 51:182-191. Microwave radiation inhibits the synthesis of DNA and increases chromosome aberrations. Garaj-Vrhovac, V., et al., Mutation Res. (1990) 243:87-93. Blocked DNA synthesis in irradiated cells and increased reproduction in uninjured cells was suggested to explain an initial decrease followed by an increase in mitotic activity of myelocaryocytes after irradiation with microwaves. Obukhan, E. I., Tsitol. Genet. (1984) 18:264-267 (translation provided).
In some studies however, the results of irradiation exposure have been less clearcut. An increase in cellular synthesis or enzyme activity has been noted. Byus, et al., Cancer Res. (1988) 48:4222-4226. Several investigators have found irradiation with microwaves has no effect on enzyme activity or DNA repair synthesis. Ward, et al., J. Microwave Power (1975) 10:315-320; Meltz, et al., Radiat. Res. (1987) 110:255-66. Other reports show both enhancement and repression of cellular enzyme activity. Dutta, S. K. and Verma, M., Curr. Sci. (1989) 58:58-63; Dutta, S. K. and Verma, M., Curr. Sci. (1988) 57:779-786.
Whether inducing or inhibitory, most of the observed microwave effects have been attributed to the increased temperature due to energy exposure, Belkhode, et al., J. Microwave Power (1974) 9:23-29, although the results of some studies indicate that thermal effects alone are not responsible for all of the consequences observed. Baranski, supra.
The syntheses, hydrolyses, and other modifications of large molecules done with microwave irradiation have utilized only simple inorganic or organic reagents such as sulfuric acid or sodium hydroxide. Furthermore, when microwave energy has been applied to drive limited, single-step chemical reactions, the results have been variable. Inconsistent temperatures, other reagents, and the absorption properties of the molecules themselves may be responsible for much of this variability. Gedye, supra; Belkhode, supra.
Although there have been reports that DNA molecules absorb microwave energy, other studies show that ions and water molecules can account for such absorption. Davis, et al., Biopolymers (1989) 28:1429-1433; Gabriel, et al., Biophys. J. (1989) 55:29-34; Maleev, et al., Biopolymers (1987) 26:1965-1970; Maleev, et al., Biopolim Kletka (1986) 2:35-38 (translation provided). As described above, investigations of DNA synthesis or repair following irradiation with microwaves have measured an increase in inhibition or damage, suggesting that DNA and the enzymes that are involved in synthesis, division, and repair are deleteriously affected by exposure to microwaves.
Recent observations of isolated DNA molecules treated with microwave energy have supported these hypotheses. Following exposure to microwave energy, plasmid DNA exhibited single-strand breaks, localized strand separations, and pseudo-restriction sites. Narasimhan, V. and Huh, W. K., Biochem. International (1991) 25:363-370. The number of single- and double-strand breaks increases in a linear relationship to both the power applied and the duration of exposure to microwaves. Sagripanti, et al., Radiat. Res. (1987) 110:219-231. No investigators have endeavored to drive enzyme-catalyzed reactions on biological molecules using microwave energy, especially large macromolecules such as DNA or RNA. In fact, microwaves appear to be avoided as an energy source for this step in macromolecule research. For example, Stroop, et al., Anal. Biochem. (1989) 182: 222-225, found that DNA could be denatured in a process using microwave energy without apparent damage, but nevertheless chose to enzymatically digest the DNA by standard procedures prior to microwave denaturation.